Pharmaceutical composition and uses thereof

ABSTRACT

Pharmaceutical compositions containing a combination of NPM inhibitor and anti-cancer agent are disclosed. Methods of inhibiting or reducing the growth of cancer cells in a subject, by administering an effective amount of nucleophosmin (NPM) inhibitor and one or more anticancer agents, whereby the symptoms and signs of cancer in the subject are reduced are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage under 35 U.S.C. 371 based on andclaiming the benefit of International Application PCT/US2013/059723,filed on Sep. 13, 2013, which claims priority from U.S. ProvisionalApplication No. 61/700,756, filed Sep. 13, 2012, the entire contents ofeach of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Nucleophosmin (NPM) is a highly conserved phosphoprotein mainly locatedin nucleoli, and shuttles between the nucleoli and cytoplasm during thecell cycle. It has been implicated in regulation of ribosome biogenesis,centrosome duplication, genome stability and apoptosis.

Cancer remains a major public health problem worldwide. It profoundlyaffects more than 1 million people in the U.S. diagnosed each year, aswell as their families and friends. Despite the advance in chemotherapyover the last 50 years, the medical community is still faced with thechallenge for treating many types of cancer. Accordingly, there is stilla need for a more effective and safe cancer treatment. The presentinvention addresses this need.

BRIEF SUMMARY OF THE INVENTION

Some embodiments provide a pharmaceutical composition comprising one ormore NPM inhibitors and one or more anti-cancer agents. Advantageously,this combination has additive or synergistic effects on cancerinhibition.

Some embodiments provide methods for reducing or inhibiting cancergrowth, comprising administering an effective amount of NPM inhibitorand an effective amount of anti-cancer agent to a subject in needthereof to thereby reduce or inhibit cancer growth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows expression of NPM and BCL2-associated X protein (BAX) inliver cancer cells following exposure to UV-B, cisplatin, ordoxorubicin. FIG. 1B shows subcellular distribution of NPM before UV-Birradiation (left panel), 3 hr after UV-B irradiation (middle panel),and 6 hr after (right panel) UVB irradiation. A subset of NPM wastranslocated to cytoplasm 6 hr after UV irradiation (indicated by anarrow in the right panel). FIG. 1C shows subcellular distribution of BAX(upper panel), mitochondria (middle panel), and BAX and mitochondria(lower panel).

FIG. 2 illustrates schematically the intracellular apoptosis and deathevasion pathways involving NPM and BAX.

FIG. 3 shows the effect of siNS (siRNA containing scrambled sequences)and siNPM (siRNA inhibits NPM expression) on liver cancer cells with orwithout treatment with UV radiation (UVB), mitomycin C (MMC),doxorubicin (DOXO) or cisplatin (CDDP).

FIG. 4 shows the effect of siNS (siRNA containing scrambled sequences),siNPM (siRNA inhibiting NPM expression), siTP53 (siRNA targeting p53)and the combination of siNPM and siTP53 on liver cancer cells with orwithout treatment with UVB, MMC, DOXO or CDDP.

FIG. 5 shows NPM expression in normal liver cells (C), liver cancercells (T) and para-liver cancer cells (N).

FIG. 6 shows NPM expression blocks the mitochondrial translocation andoligomerization of BAX in liver cancer cells following UV irradiation.FIG. 6A illustrates the expression of NPM and BAX in the cytosol and themitochondria of Mahlavu liver cancer cells following UV irradiation withor without transfected siRNA targeting NPM (NPM) or siRNA with scrambledsequences (NS). FIG. 6B illustrates the effect of siNPM and siNS on BAXdimmers (indicated with an asterisk) and BAX oligomers (twin-asterisk)in the mitochondria or the nuclei.

FIG. 7 shows effect of siNS and siNPM on liver cancer cells (Hep3B, Huh7and Mahlavu) treated with or without target cancer therapies (Sorafeniband Lapatinib).

DETAILED DESCRIPTION OF THE INVENTION Definition

As employed above and throughout the disclosure, the following terms,unless otherwise indicated, shall be understood to have the followingmeanings.

The terms “invention,” “the invention,” “this invention” and “thepresent invention” used in this patent are intended to refer broadly toall of the subject matter of this patent and the patent claims below.Statements containing these terms should be understood not to limit thesubject matter described herein or to limit the meaning or scope of thepatent claims below. Embodiments of the invention covered by this patentare defined by the claims below, not in this specification. Thespecification is not intended to identify essential features of theclaimed subject matter, nor is any portion of the specification to beused in isolation to determine the scope of the claimed subject matter.Claimed subject matter is to be understood by reference to appropriateportions of the entire specification, including all text and drawingsand each claim.

As used herein, the singular forms “a,” “an,” and “the” include theplural reference unless the context clearly indicates otherwise.

An “effective amount,” as used herein, includes a dose of an NPMinhibitor or anti-cancer agent that is sufficient to reduce the symptomsand/or signs of cancer.

The term “treating,” “treated,” or “treatment” as used herein includespreventative (e.g. prophylactic), palliative, and curative uses orresults.

The term “inhibiting” and “suppressing” includes slowing or stopping thegrowth of.

The term “subject” includes a vertebrate having or at risk of developingcancer. Preferably, the subject is a warm-blooded animal, includingmammals, preferably humans.

The term “pharmaceutically acceptable salts” of an acidic therapeuticagent of the pharmaceutical composition are salts formed with bases,namely base addition salts such as alkali and alkaline earth metalsalts, such as sodium, lithium, potassium, calcium, magnesium, as wellas 4 ammonium salts, such as ammonium, trimethyl-ammonium,diethylammonium, and tris-(hydroxymethyl)-methyl-ammonium salts.Similarly, acid addition salts, such as of mineral acids, organiccarboxylic and organic sulfonic acids, e.g., hydrochloric acid,methanesulfonic acid, maleic acid, are also possible provided to a basictherapeutic agent with a constitute such as pyridyl, as part of thestructure.

Pharmaceutical Composition

Some embodiments of the present invention is directed to pharmaceuticalcompositions for reducing or inhibiting cancer cell growth. Thepharmaceutical compositions comprising a combination of at least one NPMinhibitors and at least one anti-cancer agents. The NPM inhibitor andthe anti-cancer agent may produce additive or synergistic effects.

NPM Inhibitor

An NPM inhibitor is any agent which reduces or slows the expression ofNPM, and/or reduces NPM's activity. In one embodiment, the NPM inhibitoris (Z)-5-((N-benzyl-1H-indol-3-yl)methylene) imidazolidine-2,4-dionederivative or a pharmaceutically acceptable salt thereof. In anotherembodiment, the NPM inhibitor is 5-((N-benzyl-1H-indol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)trione derivative or a pharmaceuticallyacceptable salt thereof that incorporate a variety of substituents inboth the indole and N-benzyl moieties, which are disclosed in Sekhar etal, “The Novel Chemical Entity YTR107 Inhibits Recruitment ofNucleophosmin to Sites of DNA Damage, Suppressing Repair of DNADouble-Strand Breaks and Enhancing Radiosensitization” Clin Cancer Res2011; 17:6490-6499. In another embodiment, the NPM inhibitor is NSC348884 or a pharmaceutically acceptable salt thereof, which is disclosedin U.S. Pat. No. 8,063,089 and is incorporated herein by reference inits entirety. In another embodiment, the NPM inhibitor is CIGB-300, acyclic peptide that impairs CK2 phosphorylation after intracellulardelivery. The synthesis of CIGB-300 was described in Perea et al“Antitumor effect of a novel proapoptotic peptide that impairs thephosphorylation by the protein kinase 2 (casein kinase 2). Cancer Res2004; 64:7127-9” and is incorporated herein by reference in itsentirety. In another embodiment, the NPM inhibitor is Gambogic acid or apharmaceutically acceptable salt.

In some embodiments, the NPM inhibitor is a small interfering RNA (e.g.,siRNA, short interfering RNA or silencing RNA) targeting NPM RNAtranscription to decrease the expression of NPM. In other embodiments,the NPM inhibitor is a biosynthetic precursor of a NPM-targeted smallinterfering RNA. Small interfering RNAs are typically shortdouble-stranded RNA species with phosphorylated 5′ ends and hydroxylated3′ ends with two or more overhanging nucleotides. In some embodiments,the NPM inhibitor is an siRNA comprising s9676 (SEQ ID NOs: 2 and 3),wherein SEQ ID NO: 2 represents the sense strand and SEQ ID NO: 3represents the antisense strand. In some embodiments, the NPM inhibitoris an siRNA comprising s9677 (SEQ ID NOs: 4 and 5), wherein SEQ ID NO: 4represents the sense strand and SEQ ID NO: 5 represents the antisensestrand. In some embodiments, the NPM inhibitor is any RNA species suchas but not limited to, microRNA (miRNA), short hairpin RNA,endoribonuclease-prepared siRNA (esiRNA), natural antisense shortinterfering RNA (natsiRNA), wherein the RNA species targets the NPM RNAtranscription to decrease the expression of NPM.

In an embodiment, the NPM inhibitor is5-((N-benzyl-1H-indol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)trione(denoted as YTR107, See Formula (I)).

Anti-Cancer Agent

The anti-cancer agent includes conventional chemotherapeutic agent,target cancer therapy or radiation therapy.

The conventional chemotherapeutic agent comprises anthracyclineantibiotic, DNA synthesis inhibitor, alkylating agent, antifolate agent,metabolic inhibitor or combination thereof.

Examples of anthracycline antibiotic include, but are not limited to,doxorubicin, Epirubicin, Mitoxantrone and the like.

Examples of DNA synthesis inhibitor include, but are not limited to,mitomycin C, 5FU(5-Fluorouracil), Capecitabine, Irinotecanhydrochloride, thymitaq and the like.

Examples of alkylating agent include, but are not limited to, cisplatin,carboplatin, oxaliplatin, mitoxantrone and the like.

Examples of metabolic inhibitor include, but are not limited to,etoposide, rottlerin and the like.

Examples of antifolate agent include, but are not limited to, Nolatrexedand the like.

The target cancer therapy are medications which inhibit the growth ofcancer cells by interfering with specific targeted molecules needed forcarcinogenesis and cancer growth, rather than by simply interfering withrapidly dividing cells (e.g., with conventional chemotherapeutic agent).In some embodiments, the target cancer therapy comprises kinaseinhibitor, angiogenesis inhibitor, epidermal growth factor receptor(EGFR) inhibitor, HER2/neu receptor or the combination thereof.

Examples of kinase inhibitor include, but are not limited to, gefitinib,lapatinib, sorefenib, sunitinib, erlotinib, ABT-869, ARQ 197 and thelike.

Examples of angiogenesis inhibitor include, but are not limited to,Avastin, Brivanib, Bevacizumab, Ramucirumab and the like.

Examples of EGFR inhibitor include, but are not limited to, Gefitinib,Cetuximab and the like.

Examples of HER2/neu receptor include, but are not limited to,Trastuzumab, Lapatinib, or the like.

Anti-cancer agents are known for side effects, such as weight loss, lossof hair, anemia, neutropenia and thrombocytopenia. These side effectsmay be overcome by administering lower dosage of anti-cancer agent incombination with one or more NPM inhibitors to achieve the desiredtherapeutic effect. The observed synergistic or additive effect of apharmaceutical composition comprising a combination of a NPM inhibitorand an anti-cancer agent (e.g., Cisplatin) may afford effectiveinhibition or reduction of cancer cell growth wherein one or even all ofthe lower dosages of the anti-cancer agents would not be sufficient tohave a therapeutic effect when the respective anti-cancer agent is usedin monotherapy.

The pharmaceutical compositions to be administered according to themethods of some embodiments provided herein can be readily formulatedwith, prepared with, or administered with, a pharmaceutically acceptablecarrier. Such pharmaceutical compositions may be prepared by varioustechniques. Such techniques include bringing into association activecomponents (such as an NPM inhibitor) of the pharmaceutical compositionsand a pharmaceutically acceptable carrier. In one embodiment,pharmaceutical compositions are prepared by uniformly and intimatelybringing into association active components of the pharmaceuticalcompositions with liquid carriers, with solid carriers, or with both.Liquid carriers include, but are not limited to, aqueous formulations,non-aqueous formulations, or both. Solid carriers include, but are notlimited to, biological carriers, chemical carriers, or both.

The pharmaceutical compositions are administered in an aqueoussuspension, an oil emulsion, water in oil emulsion andwater-in-oil-in-water emulsion, and in carriers including, but notlimited to, creams, gels, liposomes (neutral, anionic or cationic),lipid nanospheres or microspheres, neutral, anionic or cationicpolymeric nanoparticles or microparticles, site-specific emulsions,long-residence emulsions, sticky-emulsions, micro-emulsions,nano-emulsions, microspheres, nanospheres, nanoparticles and minipumps,and with various natural or synthetic polymers that allow for sustainedrelease of the pharmaceutical composition including anionic, neutral orcationic polysaccharides and anionic, neutral cationic polymers orcopolymers, the minipumps or polymers being implanted in the vicinity ofwhere composition delivery is required. Furthermore, the activecomponents of the pharmaceutical compositions provided herein are usefulwith any one, or any combination of, carriers. These include, but arenot limited to, anti-oxidants, buffers, and bacteriostatic agents, andoptionally include suspending agents, thickening agents orpreservatives.

For administration in a non-aqueous carrier, active components of thepharmaceutical compositions provided herein are emulsified with amineral oil or with a neutral oil such as, but not limited to, adiglyceride, a triglyceride, a phospholipid, a lipid, an oil andmixtures thereof, wherein the oil contains an appropriate mix ofpolyunsaturated and saturated fatty acids. Examples include, but are notlimited to, soybean oil, canola oil, palm oil, olive oil and myglyol,wherein the number of fatty acid carbons is between 12 and 22 andwherein the fatty acids can be saturated or unsaturated. Optionally,charged lipid or phospholipid is suspended in the neutral oil. Asuitable phospholipid is, but is not limited to, phosphatidylserine,which targets receptors on macrophages. The pharmaceutical compositionsprovided herein are optionally formulated in aqueous media or asemulsions using known techniques.

The pharmaceutical compositions provided herein may optionally includeactive agents described elsewhere, and, optionally, other therapeuticingredients. The carrier and other therapeutic ingredients must beacceptable in the sense of being compatible with the other ingredientsof the composition and not deleterious to the recipient thereof.

The pharmaceutical compositions are administered in an amount effectiveto inhibit or reduce cancer cell growth. The dosage of thepharmaceutical composition administered will depend on the severity ofthe condition being treated, the particular formulation, and otherclinical factors such as weight and the general condition of therecipient and route of administration.

Useful dosages of the pharmaceutical compositions provided herein aredetermined by comparing their in vitro activity, and in vivo activity inanimal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known in the art; for example,see U.S. Pat. No. 4,938,949, which is incorporated by reference herein.

The NPM inhibitor or the anti-cancer agent can be administered at anyeffective amount. In some embodiments, they may be administered at adose ranging from about 0.01 μg to about 5 g, from about 0.1 μg to about1 g, from about 1 μg to about 500 mg, from about 10 μg to about 100 mg,from about 50 μg to about 50 mg, from about 100 μg to about 10 mg, fromabout 0.5 μg to about 5 μg, from about 15 μg to about 500 μg, from about3 μg to about 1 mg, from about 7 μg to about 1 mg, from about 10 μg toabout 20 μg, from 15 μg to about 1 mg, from about 15 μg to about 300 μg,from about 15 μg to about 200 μg, from about 15 μg to about 100 μg, fromabout 15 μg to about 60 μg, from about 15 μg to about 45 μg, from about30 μg to about 60 μg, or from about 50 μg to about 100 μg. In certainembodiments, the NPM inhibitor or the anti-cancer agent is administeredin a dose ranging from about 0.1 μg/kg bodyweight to about 200 mg/kgbodyweight, from about 1 μg/kg bodyweight to about 100 mg/kg bodyweight,from about 100 μg/kg to about 50 mg/kg bodyweight, from about 0.5 mg/kgto about 20 mg/kg bodyweight, from about 1 mg/kg to about 10 mg/kgbodyweight, from about 10 μg/kg bodyweight to about 200 μg/kgbodyweight, at least about 0.01 μg/kg bodyweight, about 0.1 μg/kgbodyweight, or at least about 0.5 μg/kg bodyweight.

In accordance with the methods provided herein, the pharmaceuticalcomposition is delivered by any of a variety of routes including, butnot limited to, injection (e.g., subcutaneous, intramuscular,intravenous, intra-arterial, intraperitoneal, intradermal); cutaneous;dermal; transdermal; oral (e.g., tablet, pill, liquid medicine, ediblefilm strip); implanted osmotic pumps; suppository, aerosol spray,topical, intra-articular, ocular, nasal inhalation, pulmonaryinhalation, impression into skin and vaginal.

The pharmaceutical composition may be administered in a single dosetreatment or in multiple dose treatments, over a period of timeappropriate to the condition being treated. The pharmaceuticalcomposition may conveniently be administered at appropriate intervals,for example, once a day, twice a day, three times a day, once everysecond day, once every three days or once every week, over a period ofat least 3 months or until the symptoms and signs of the conditionresolved.

The Method of Suppressing Cancer Growth

Some embodiments of the invention is directed to methods of inhibitingor suppressing cancer growth in a subject, which comprises theadministration an effective amount of at least one NPM inhibitor and atleast one anti-cancer agent (as described herein) to a subject in needthereof, whereby the symptoms and/or signs of the cancer in the subjectare reduced.

Nucleophosmin or NPM (SEQ ID NO:1) is a highly conserved anti-apoptosisprotein that shuffles between the nucleoli and cytoplasm during the cellcycle. Under normal condition, NPM located in the nucleoli, but a smallamount is present in the nucleoplasm (FIG. 2B, left). BCL2-associated Xprotein (BAX), a mitochondria mediated apoptosis protein, is mainlylocated in the nucleoplasm, but a small amount is present in the cytosol(FIG. 1C, left).

In response to cell stress (e.g., UV radiation or contacting withanti-cancer agents), NPM is translocated from the nucleolus tonucleoplasm (FIG. 2B, middle panel) and cytosol (FIG. 2B, right panel),and bound to BAX. Without being bound by any particular theory, it isbelieved that the binding of NPM to BAX in the cytosol effectivelyblocks mitochondrial translocation, oligomerization and activation ofBAX, thereby rendering cells resistant to cell death (see death evasionpathway in FIG. 2).

By inhibiting NPM expression, cytosolic BAX is translocated tomitochondria and targeted the mitochondrial inner membrane, where BAX isoligomerized. The mitochondria forms pores, loses membrane potential,releases cytochrome C into cytoplasm, and activates cascades forapoptosis (see apoptosis pathway in FIG. 2).

The present compositions and methods can be used to treat or inhibit thegrowth of any type of cancer. In some embodiments, the cancer to betreated or the cancer growth to be inhibited is a solid or hematologicaltumor, such as, for example, liver, bile duct, breast, lung, gastric,pancreatic, colorectal, uterus, cervical cancer, leukemias andlymphomas.

Treatment may be administered alone, or as an adjuvant to surgery, e.g.,before surgery to reduce the tumor size and/or following surgery toreduce the possibility of metastases, e.g., by inhibition of the growthand migration of circulating tumor cells through the blood stream.

The NPM inhibitor can be administered before, after or simultaneouslywith the anti-cancer agent.

In certain instances, the therapy includes a combination of anti-canceragents to be administered together with an NPM inhibitor.

The following examples further illustrate the present invention. Theseexamples are intended merely to be illustrative of the present inventionand are not to be construed as being limiting.

Material and Methods

1. Preparation of Cancer Cells, NPM Expression Inhibitors andTransfection

Human hepatocarcinoma (HCC) lines, HepG2 (wild-type p53), Hep3B(null-genotype p53), Huh7 (C200Y mutated p53), Mahlavu (S249R mutatedp53), colorectal cancer cell line HCT-116, ovarian cancer cell linesSKOV3 and MDAH2774, lung cancer cell line A549, cervical cancer cellline HeLa and breast cancer cell line MCF7 were obtained from AmericanType Culture Collection (Manassas, Va.). Gastric carcinoma cell lineTSGH was purchased from University of California, San Francisco (SanFrancisco, Calif.), cholaniocarcinoma cell line HuCCT-1 was purchasedfrom JCRB cell bank and uterus cancer cell line Ishikawa was purchasedfrom Sigma-Aldrich (Switzerland).

Pre-designed small interference RNAs (siRNAs) targeting NPM (see SEQ IDNOs:2-5) and p53 (siTP53), and siRNAs with scrambled sequences (siNS)were purchased from Ambion, Austin, Tex. In particularly, the siNM usedin the study was the Silencer® Select Negative Control #1. Transfectionwas performed as previously described in Hsieh et al, “Identifyingapoptosis-evasion proteins/pathways in human hepatoma cells viainduction of cellular hormesis by UV irradiation.” J Proteome Res 2009;8:3977-3986.

NPM inhibitor NSC348884 was purchased from SantaCruz Biotechnology(Santa Cruz, Calif.) and Gambogic acid was purchased from and Enzo LifeSiences (Farmingdale, N.Y.).

2. UV Irradiation, Drug Treatments, and Cell Survival/Viability Assays

In Example 1, 1×10⁴ cancer cells were seeded into each well of a 96-wellplate followed by transfection with siRNAs in Example 1. Forty-eighthours after transfection, cells at 90% confluence were treated with (50mJ/cm²) of UV-B (290-320 nm) or one of the following chemotherapeuticagents: Mitomycin C (Kyowa Hakko Kogyo Co., Ltd.), cisplatin(Bristol-Myers Squibb S.R.L.) or doxorubicin (Pfizer Italia S.R.L.).Target cancer therapy, such as Sorafenib (kindly provided by BayerHealthCare, German) and Lapatinib (purchased from GlaxoSmithKline plc)were prepared in DMSO. Solvent was added to untreated HCC cells ascontrol in each experiment. Cell viability was assessed at 24 h to 48 hafter treatment.

For UV irradiation group, cell viability/survival was determined by XTTassay (Roche Applied Science, Mannheim, Germany) 24 h after the exposureto 30, 65, or 100 mJ/cm² of UV-B. The experiments were conducted atleast twice in triplicate and the mean of each dose was used tocalculate the half maximal inhibitory concentration (IC₅₀).

In Example 6, 2×10⁴ cancer cells were seeded into each well of a 24-wellplate, cultured overnight followed by combination drug treatment. IC50,IC90, cell viability and Combination Index were assessed 72 hours afterexposure.

3. In Situ Proximity Ligation Assays and Co-Immunoprecipitation

Anti-NPM mouse monoclonal antibody and anti-BAX rabbit polyclonalantibody or anti-actin rabbit polyclonal antibody (negative control)were used as primary antibodies and anti-mouse and anti-rabbitantibodies coupled with short complementing DNA strands were used assecondary antibodies. Ligation of the DNA strands to a circularizedoligo in case of direct contact between NPM and BAX, and the subsequentrolling circle amplification incorporating labeled nucleotides wasperformed using the Duolink II kit (Olink Bioscience, Uppsala, SWE)according to the manufacturer's instruction. After being washed andcounterstained with DAPI (4′,6-diamidino-2-phenylindole, a fluorescentstain for DNA), the slides were mounted and inspected under thefluorescence microscope.

Cells were grown in a 10 cm plate for co-immunoprecipitation (co-IP).500 iul of co-IP lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA,1% TritonX-100, pH7.4, 1 mM PMSF, 1 mM Na3VO4, 1 ug/ml aprotinin) wasadded while the culture dish was placed on ice. Cells were scraped andthen lyzed by gentle rocking on ice for 15 minutes. Cellular lysate wascentrifuged for 5 min at 12000 g at 4° C. to remove debris. Thesupernatant was collected in a fresh tube and 2 ug of the first antibodyagainst either NPM or BAX was added. The reaction mixture was rockedgently overnight at 4° C., and followed by the addition of 20 ul of 50%slurry of protein A-sepharose beads. The resultant mixture was incubatedat 4° C. for 2 hours followed by centrifugation at 6000 g for 5 min at4° C. The supernatant was kept as IP efficiency control, while the beadswere washed three times with a buffer (10 mM Tris-HCl, 500 mM NaCl, pH7.4) and heated in 50 ul of 2×SDS loading buffer at 95° C. for 10minutes, followed by immunoblotting with the identified antibodies asdescribed above.

4. Patients and Tissue Samples

The Internal Review Board for Medical Ethics of Chang Gung MemorialHospital in Taiwan approved the specimen collection procedures. HCC andits surrounding tissues, as well as the related clinical data from 90HCC patients were obtained from the Taiwan Liver Cancer Network (TLCN).All HCC tissues were examined and the two core samples from mostrepresentative areas in the tissue samples were selected for the tissuemicroarray blocks. Two core samples were selected from different areasof each HCC tissues.

The ImmunoHistoChemistry (IHC) scores were determined by two independentobservers. If there was disagreement between the two observers, theslides were re-examined and a consensus was reached by the observers.IHS Score 0 indicates negative result, 1 indicates weakly positiveresult, 2 indicates positive result and 3 indicates strongly positiveresult.

The combined effect of the drugs was analyzed and expressed asCombination Index (CI), using the method of T. C. Chou: TheoreticalBasis, Experimental Design, and Computerized Simulation of Synergism andAntagonism in Drug Combination Studies, Pharmacological Reviews. 2006;58(3):621-81, the entire disclosure of which is incorporated herein byreference. CI between 0.3-0.7 indicates synergism, 0.7-0.85 indicatesmoderate synergism, 0.85-0.9 indicates slight synergism, 0.9-1.1indicates additive, 1.1-1.2 indicates slight antagonism, 1.2-1.45indicates moderate antagonism and 1.45-3.3 indicates antagonism.

Tumor staging was determined in accordance with tumor-node-metastasis(TNM) staging system, as suggested by the American Joint Committee onCancer/International Union Against Cancer.

The Chi-square test and Fishers Exact test were used for comparisonbetween variables. Kaplan-Meier analysis and the log-rank test were usedto illustrate recurrence-free and overall survival probability afterpatients received primary curative hepatectomy.

Example 1 In-Vitro Evaluation of NPM Inhibitor and Anti-Cancer AgentsCombination in HCC

HCC cell lines with different p53 backgrounds, including HepG2(wild-type p53), Huh7 (C200Y mutated p53), Mahlavu (R249S mutant p53),and Hep3B (deleted p53) were used in this study.

Referring to FIG. 3, the doses for UVB, MMC, DOXO and CODP at 0 mJ/cm²or mg/ml indicate HCC cells were not treated with UV-B or conventionalchemotherapeutic agents. The siNPM (siRNA inhibiting NPM expression)bars represents groups without UV-B or chemotherapeutic agent treatmentbut with NPM suppression. The siNS bars represent groups without UV-B,chemotherapeutic agent treatment or NPM suppression. Inhibition of NPMexpression by siNPM was confirmed by immunoblotting (FIG. 3, right lowerpanel).

HCC cells were treated with UV-B or one of the conventionalchemotherapeutic agents when the doses for UVB, MMC, DOXO and CODP areabove 0 mJ/cm² or ug/ml. In this group, the siNs bars represent groupswithout NPM expression inhibition, but were treated with UV-B orconventional chemotherapeutic agents. The siNPM bars groups with NPMsuppression and treatment with a chemotherapeutic agent or UV-Bradiation. Cell viability was measured by XTT assay. * (p<0.05) and **(p<0.01) indicate statistical significance between cells transfectedwith siNPM and with siNS.

NPM expression inhibition in combination with chemotherapy or UV-Btreatment significantly reduced cell viability of HCC cells compare toNPM expression inhibition alone. The results show that the combinationof NPM expression inhibition and chemotherapy or UV-B treatment iseffective in HCC treatment.

Referring to FIG. 7, the doses for sorefenib and lapatinib at 0 uM or nMindicate HCC cells were not treated with target cancer therapy. At 0 uMor nM, the siNS (black) bars represent no NPM expression inhibition andno target cancer therapy (control group), while the siNPM (grey) barsrepresent groups with NPM expression inhibition, but without targetcancer therapy.

The doses for sorefenib and lapatinib above 0 uM or nM indicate HCCcells were treated with target cancer therapy. siNS (black) bars withsorefenib and lapatinib doses above 0 uM or nM represent groups withoutNPM expression inhibition, but were treated with target cancer therapy,whereas siNPM (grey) bars with sorefenib and lapatinib doses above 0 uMor nM represent groups with NPM expression inhibition and treated withtarget cancer therapy.

Inhibiting NPM expression in combination with target cancer therapysignificantly enhanced the cellular susceptibility in Huh7, Hep3B andMahlavu cells compare to NPM expression suppression or target cancertherapy alone. The results show that the combination of NPM expressionsuppression and target cancer therapy provide synergistic effect in HCCtreatment.

The role of p53 in death evasion orchestrated by NPM in cancer cells wasfurther evaluated. The expression of NPM, p53, or simultaneously NPM andp53 were silenced by siNPM and siTP53 (FIG. 4). Silencing of p53expression alone did not significantly change the sensitivity of thetreatments in Huh7, Hep3B, and Mahlavu cells (FIG. 4, siTP53 vs. siNS).Simultaneous silencing of p53 and NPM did not further alter thesensitizing effect exerted by silencing of NPM alone [FIG. 4, siNPM/siNSvs. (siNPM+siTP53)/siNS]. NPM apparently executes its death-evasionactivity independently of p53. These findings are of great clinicalsignificance, since p53 mutations are found in more than half of humancancers including HCC, especially in advanced stage HCC.

Example 2 Induction of NPM and BAX Expression by Cellular Stresses

Now referring to FIG. 1A, NPM was upregulated in Huh7, Hep3B, andMahlavu cells following UV-B (50 mJ/cm²), cisplatin [5.5, 69, and 6.4μg/ml for Hep3B (3B), HepG2 (G2) and Mahlavu (ML), respectively], anddoxorubicin [1.4, 8.8, and 5 μg/ml for Hep3B, HepG2 and Mahlavu,respectively] exposure. BAX expression was also increased in all threeHCC cell lines following the treatment of UV-B, cisplatin, anddoxorubicin. The expression of β-actin was used as the loading control.Simultaneous induction of BAX (pro-apoptosis) and NPM (anti-apoptosis)of cells upon cellular stress represents counteracting mechanismsregulating apoptosis versus survival response.

Example 3 Nucleoplasmic and Cytoplasmic Translocation of NPM FollowingCellular Stresses

Prior to UV irradiation, NPM was mainly located in the nucleoli and asmall amount was present in nucleoplasm (FIG. 1B, left panel), while BAXwas primarily located in nucleoplasm and a small amount was located inthe cytoplasm (FIG. 1C, left panel). Following UV irradiation, NPM wastranslocated from nucleoli to nucleoplasm (FIG. 1B, middle), andcytoplasm (FIG. 1B, right panel, indicated by an arrow). On the otherhand, BAX was translocated to cytosol and accumulated in themitochondria, particularly in cells undergo apoptosis (FIG. 1C, rightpanel; indicated by arrows).

Following the suppression of NPM expression by siRNA, HCC cells withrelatively low NPM expression have more BAX aggregated in themitochondria and were found to be more prone to apoptosis, whereas cellswith relatively high NPM level have less mitochondrial BAX accumulationand were found to be more resistance to apoptosis. These findingssuggest that the anti-apoptosis activity of NPM involves the blockade ofBAX mitochondrial translocation.

Example 4 Blockade of BAX Mitochondria Translocation and Oligomerizationby NPM

FIG. 6A illustrates cytoplasmic NPM increased after UV irradiation,whereas BAX increased in the cytosol and mitochondria after UVirradiation. Suppressing NPM expression by siRNA reduced the cytosolicBAX, while mitochondrial BAX level increased. This suggests BAXmitochondrial translocation was blocked by NPM in response to cellularstress such as UV treatment. Prohibitin (PHB) and glyceraldehyde3-phosphate dehydrogenase (GAPDH) were used as the markers formitochondrial and cytosolic components, respectively. Similar result formitochondrial BAX enhancement was observed by inhibiting NPM withchemotherapeutic agents, such as staurosporin in Hep3B and Mahlavucells.

A non-reducing condition for preparing cellular proteins was employed tovalidate the above findings. Inhibiting NPM expression (FIG. 6B, lane 2)greatly increased the dimmers and oligomers of mitochondrial BAXfollowing UV irradiation, whereas the BAX dimmers and oligomers werebarely detected in the mitochondria before UV irradiation (FIG. 6B,lane 1) or without NPM expression inhibition (FIG. 6B, lane 3). Inconclusion, NPM blocks the mitochondrial translocation andoligomerization of BAX in HCC cells.

Example 5 Upregulation of NPM in Human HCC was Associated with HepatitisB, Portal Vein Invasion, High Recurrence and Poor Prognosis

Using immunoblotting assay, NPM level was found to be high in 4 out of 6HCC samples compared to the matched para-HCC liver tissues and a normalliver tissue (FIG. 5).

The expression of NPM in 90 pairs of HCC and para-HCC liver samples wereexamined. Overexpression of NPM was found in 38.9% (35/90) of HCCsamples and strongly associated with chronic hepatitis B (p<0.0001),advanced cancer stages (p=0.0015), portal vein invasion (p<0.001), tumorrecurrence (p=0.0148), and poor overall survival (p=0.0229). See Tables1-4. NPM upregulation is associated with higher tumor recurrence andlower overall survival, as demonstrated via Kaplan-Meier analyses andlog-rank test.

TABLE 1 Correlation of NPM expression to clinical manifestations ofpatients with HCC IHC score statistics 0 >0 p test Etiology HBV 18 27<0.001 Fisher's exact HCV 37 8 Age* mean ± 59.3 ± 3.8 56.8 ± 3.6 0.539Student's t SEM Gender* male 12 16 0.428 Fisher's exact female 6 11Cirrhosis* yes 10 22 0.146 Fisher's exact no 8 5 AFP* >400 12 19 0.724Fisher's exact <400 6 8 Tumor I 6 9 0.0015 Spearman stage* II 9 6correlation III 3 12 Vascular yes 2 23 <0.001 Fisher's exact invasion*no 16 4 Recurrence* 0.0148 Log rank Overall 0 vs. >0 0.1101 Log ranksurvival* 0, 1, 2 vs. 3 0.0229 Log rank A total of 90 cases with HCCincluding 45 cases with chronic hepatitis B and 45 cases with chronichepatitis C were included and assayed on tissue arrays byimmunohistochemistry (IHC) IHC scores were determined by two independentpathologists. IHC score: 0, negative; 1, weakly positive; 2, positive;3, strongly positive HBV: chronic hepatitis B; HVC: chronic hepatitis C*Only the 45 cases with HBV-related HCC were included for analyses.

TABLE 2 Immunohistochemistry scores for NPM expression in 45 pairs ofhepatitis B-related HCC and non-HCC liver tissues on tissue arrays. IHCIHC IHC IHC score 0 score 1 score 2 score 3 subtotal Stage I 6 5 2 2 15Stage II 9 0 6 0 15 Stage III 3 5 1 6 15 subtotal 18 10 9 8 45

TABLE 3 Immunohistochemistry scores for NPM expression in 45 pairs ofhepatitis C-related HCC and non-HCC liver tissues on tissue arrays. IHCIHC IHC IHC score 0 score 1 score 2 score 3 subtotal Stage I 11 3 1 0 15Stage II 12 0 2 1 15 Stage III 14 1 0 0 15 subtotal 37 4 3 1 45

TABLE 4 Correlation of NPM expression to clinical manifestations ofpatients with HCC IHC score P value HBV vs HCV 0, 1, 2, 3 <0.0001^(a) 0vs. 1, 2, 3 0.0001 0, 1 vs. 2, 3 0.0028 0, 1, 2 vs. 3 0.03 Stages (HBVonly) 0, 1, 2, 3 0.0015^(b) 0 vs, 1, 2, 3 0.0821 0, 1 vs. 2, 3 0.5159 0,1, 2 vs. 3 0.0174 Portal-vein invasion 0 vs. 1, 2, 3 0.0019 (HBV only)0, 1 vs. 2, 3 0.0075 Disease-free survival 0 vs. 1, 2, 3 0.0148^(c) (HBVonly) 0, 1, 2 vs. 3 0.1193^(c) Overall survival 0 vs. 1, 2, 3 0.1101^(c)(HBV only) 0, 1, 2 vs. 3 0.0229^(c) HBV = Chronic Hepatitis B; HCV =Chronic Hepatitis C; ^(a)Kruskal-Wallis test; ^(b)Spearman correlation =0.229; ^(c)Log-rank test.

Example 6 In-Vitro Evaluation of NPM Inhibitor and Anti-Cancer AgentCombination in Various Cancer Cell Lines

Combinations of NPM inhibitor and an anti-cancer agent were evaluated inthe following cancer cell lines: HCC cell lines (Huh7 and Mahlavu),gastric carcinoma cell line (TSGH), cholangiocarcinoma cell line(HuCCT-1), colorectal carcinoma cell line (HCT-116), ovarian cancer celllines (SKOV3 and MDAH2774), lung cancer cell (A549), uterine cancer cellline (Ishikawa), cervical cancer cell line (HeLa) and breast cancer cellline (MCF7).

As shown in Table 5, the overall effect of Sorafenib (an anti-canceragent) and NPM inhibitor (NSC348884 or Gambogic acid) combination on HCCcell line indicates additivity to synergy.

TABLE 5 The effect of Sorafenib in combination with NSC348884 orGambogic acid on HCC. Combination Indices (CI) Drug 50% 75% 90% (MolarCell Effective Effective Effective Overall Ratio) line Dose Dose Doseresult Sorafenib + Huh7 1.08 1.06 1.18 Additive NSC348884 (1:1)Sorafenib + Huh7 0.52 0.68 1.01 Synergism Gambogic at 50% acid and 75%,(1:2) additive at 90% Sorafenib + Mahlavu 0.72 0.83 0.96 SynergismGambogic at 50% acid and 75%, (2:1) additive at 90%

The effect of Lapatinib (an anti-cancer agent) and Gambogic acid (an NPMinhibitor) combination on eight different cancer cell lines aresummarized in Table 6. Taken as a whole, the results of all cancer celllines indicate additivity to synergy.

TABLE 6 The effect of Lapatinib in combination with Gambogic acid onvarious cancer cell lines. Combination Indices (CI) 50% 75% 90% CancerCell Effec- Effec- Effec- Lapatinib:Gambogic line tive tive tive OverallAcid Molar Ratio (Origin) Dose Dose Dose result 15:2 Huh7 0.84 0.88 1.05Synergism (HCC) at 50% and 75%; additive at 90%  2:1 Mahlavu 0.67 0.750.85 Synergism (HCC) 15:2 TSGH 0.94 0.90 0.87 Synergism (Gastric) at90%, additive at 50% and 75% 15:2 HuCCT-1 0.69 0.75 0.81 Synergism(Cholangio Carcinoma) 15:2 HCT-116 0.71 0.66 0.62 Synergism (Colorectal)15:2 SKOV3 0.67 0.54 0.47 Synergism (Ovarian) 15:1 MDAH2774 0.89 0.890.93 Synergism (Ovarian) at 50% and 75%, additive at 90% 15:2 A549 0.971.02 1.01 Additive (lung) 10:1 Ishikawa 0.77 0.79 0.79 Synergism(Uterus)  2:1 HeLa 0.73 0.64 0.56 Synergism (Cervical) 15:1 MCF7 0.980.95 0.96 Additive (Breast)

The effect of Lapatinib (an anti-cancer agent) and NSC348884 (an NPMinhibitor) combination on eight different cancer cell lines aresummarized in Table 7. Taken as a whole, the results of all cancer celllines indicate mixed additivity/synergy.

TABLE 7 The effect of Lapatinib in combination with NSC348884 on variouscancer cell lines. Combination Indices (CI) 50% 75% 90% Cancer CellEffec- Effec- Effec- Lapatinib:NSC348884 line tive tive tive OverallMolar Ratio (origin) Dose Dose Dose result 15:4 Mahlavu 1.74 1.79 1.87Antag- (HCC) onism 20:3 TSGH 0.14 0.35 0.90 Syner- (Gastric) gism 20:3HuCCT-1 — 0.17 0.25 Syner- (Cholangio gism carcinoma) 15:2 HCT-116 0.630.77 0.94 Syner- (Colorectal) gism 15:4 SKOV3 0.96 0.94 0.93 Addi-(Ovarian) tive  5:1 MDAH2774 0.32 0.40 0.50 Syner- (Ovarian) gism 15:2A549 0.91 0.91 0.95 Addi- (Lung) tive 20:1 Ishikawa 1.09 1.06 1.04 Addi-(Uterus) tive 15:4 HeLa 1.65 1.71 1.77 Antag- (Cervical) onism  5:1 MCF70.49 0.86 — Syner- (Breast) gism

These results demonstrate that combination treatment of Lapatinib (ananti-cancer agent) with NSC348884 (an NPM inhibitor) yield additive tosynergistic anti-cancer effect in most cancer cell lines, except Mahlavu(HCC) and HeLa (cervical) cell lines.

Referring to Table 8, lower concentrations of Lapatinib and NSC348884are needed for inhibiting HCC and cervical cancer in combination therapyform than that of monotherapy form. The results indicate Lapatinib andNSC348884 combination is effective in cancer cell inhibition and can bepresent in a dose that is less than to the dosage normally administeredin monotherapy regimen.

TABLE 8 IC50 of Lapatinib and NSC348884 as monotherapy and combinationtherapy in HCC and cervical cancer cell IC50 (μM) Cancer cell LineMonotherapy Combination Therapy (Origin) Lapatinib NSC348884 LapatinibNSC348884 Mahlavu 12.83 6.15 14.4 3.84 (HCC) HeLa 20.76 6.24 18.45 4.92(Cervical)

What is claimed is:
 1. A method for reducing or inhibiting cancer cellsin a subject, the method comprising the steps of: (a) contacting thecancer cells with an NPM inhibitor, wherein the NPM inhibitor isgambogic acid; and (b) administering an effective amount of ananti-cancer agent, wherein the anti-cancer agent is target cancertherapy, wherein said target cancer therapy is “sorefenib or lapatinib”;wherein the cancer cells are selected from the group consisting ofhepatocarcinoma, gastric cancer, cholangiocarcinoma, colorectal cancer,ovarian cancer, lung cancer, uterine cancer, and breast cancer, whereinsaid steps of contacting and administering provide a synergistic effecton reducing or inhibiting said cancer cells in said subject.
 2. Themethod of claim 1, wherein the NPM inhibitor is administered prior to,after or simultaneously with the anti-cancer agent.
 3. The method ofclaim 1, wherein the ratio of the anti-cancer agent to NPM inhibitor is20:1 to 2:1.